Method of quantitative assay for vitamin B12 and reagent for assaying vitamin B12

ABSTRACT

The present invention is directed to a method of quantitative assay for a vitamin B 12  -containing substance which comprises culturing marine methanol-utilizing bacteria having B 12  auxotrophy in a medium for quantitative assay of the bacteria containing pyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acid ester and, quantitatively determining vitamin B 12  in the vitamin B 12  -containing substance as a function of the degree of growth thereof and, to a reagent for the B 12  -containing substance comprising a kit for combination of an ampule or vial having sealed therein a dry viable bacteria composition of a marine methanol-utilizing bacteria for inoculation and an ampule or vial having sealed therein a medium for the bacteria containing pyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acid ester, or in addition thereto, further an ampule or vial having sealed therein a standard dilution of vitamin B 12  having a serial concentration.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of quantitative microorganismassay for a vitamin B₁₂ (hereafter simply referred to as B₁₂)-containingsubstance and a reagent used for the method of quantitative assay. Moreparticularly, the present invention relates to a method of quantitativeassay of B₁₂ in a B₁₂ -containing substance using marinemethanol-utilizing bacteria requiring B₁₂ for growth and capable ofassimilating methanol quickly in a simple manner and a reagent forquantitative assay of the B₁₂ -containing substance comprising acomposition of dry viable bacteria for inoculation of marinemethanol-utilizing bacteria having B₁₂ auxotrophy used for thequantitative assay and a liquid medium containing methanol and alsocontaining pyrroloquinoline-quinone and a polyoxyethylene sorbitan fattyacid ester and a B₁₂ standard solution.

2. Brief Description of the Prior Art

Physicochemical methods and microbiological methods have been hithertoknown as quantitative assay for B₁₂ -containing substances, i.e.,substances containing vitamin B₁₂. Among the microbiological methods, amethod of quantitatively determining B₁₂ in B₁₂ -containing substancesusing marine methanol-utilizing bacteria requiring B₁₂ for growth isknown to be excellent (Published Unexamined Japanese Patent ApplicationNo. 19022/1980).

When an attempt is made to quantitatively determine B₁₂ in B₁₂-containing substances, however, this method is disadvantageous in thatbacteria do not necessarily have stable growth and the B₁₂ cannot bequantitatively determined quickly with accuracy.

Problems to be solved by the present invention are in the development ofan improved method for quantitatively assaying a B₁₂ -containingsubstance that eliminates the defects possessed by conventional assaymethods of B₁₂ -containing substances using marine methanol-utilizingbacteria having B₁₂ auxotrophy, namely, failing to quantitativelydetermine the B₁₂ -containing substance stably and quickly with goodaccuracy, and reagents for quantitative assay for B₁₂.

As a result of various investigations to solve the foregoing problems,the present inventors have found that by supplementingpyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acid esterto a medium for quantitative assay using marine methanol-utilizingbacteria having B₁₂ auxotrophy, the B₁₂ in a B₁₂ -containing substancecan be quantitatively determined stably with accuracy. The presentinvention has been accomplished based on this finding.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a quick and simplemethod of quantitative assay for B₁₂ -containing substances, i.e.,substances containing vitamin B₁₂, using marine methanol-utilizingbacteria.

Another object of the present invention is to provide a reagent used ina quantitative assay for B₁₂ -containing substances using marinemethanol-utilizing bacteria.

The method of quantitative assay for a vitamin B₁₂ -containing substanceaccording to the present invention is characterized by culturing marinemethanol-utilizing bacteria having B₁₂ auxotrophy in a medium forquantitative assay for the bacteria containing pyrroloquinoline-quinoneand a polyoxyethylene sorbitan fatty acid ester, and quantitativelydetermining vitamin B₁₂ depending upon degree of growth thereof.

The present invention is also directed to a reagent for quantitativeassay used in microbiological assay for a B₁₂ -containing substance,comprising a kit for combination of an ampule or vial having sealedtherein a dry viable bacteria composition of marine methanol-utilizingbacteria having B₁₂ auxotrophy for inoculation and an ampule or vialhaving sealed therein a medium for quantitative assay for the bacteriacontaining pyrroloquinoline-quinone and a polyoxyethylene sorbitan fattyacid ester, or further an ampule or vial having sealed therein astandard dilution of vitamin B₁₂ having a serial concentration.

DETAILED DESCRIPTION OF THE INVENTION

As microorganisms which can be used in accordance with the presentinvention, mention may be made of Alteromonas thalassomethanolica YK4007, FERM BP-1401 (This strain deposited under FERM P-3621 on June 22,1976 at Fermentation Research Institute, Agency of Industrial Scienceand Technology, 1-3, Higashi, 1-chome, Yatabe-machi, Tsukuba-gun,Ibaraki-ken, Japan and Transferred on July 2, 1987 to Internationaldeposition FERM BP-1401 under the treaty on the InternationalRecognition of the Deposit of Microorganisms for the purpose of patentprocedure. Its bacteriological properties are described in J. Ferment.Technol., 58, 99-106 (1980)) which has already been separated as amarine methanol-utilizing bacteria based on characteristics that thebacteria can be well assimilated using methanol as only one carbonsource and seawater can be utilized as water for the medium, etc.; andthe like. All of these microorganisms are characterized by requiring B₁₂for growth.

As media for quantitatively assaying the B₁₂ -containing substance thatcan be used in the present invention, conventional media forquantitatively assaying marine methanol-utilizing bacteria having B₁₂auxotrophy heretofore used are usable except for that containingpyrroloquinoline-quinone and the polyoxyethylene sorbitan fatty acidester. Additional incorporation of iron sulfates, zinc sulfates, sodiumthiosulfate, thioglycolic acid, etc. in the media having the aforesaidcomposition for quantitative assay give good assay results on someoccasions.

It is desired that the amount of pyrroloquinoline-quinone to beincorporated is in the range of 10 to 60 ng per 100 ml of the medium forquantitative assay; when the amount exceeds 75 ng, good assay resultscannot always be obtained because the growth of the microorganism isinhibited. Further when the amount is less than 10 ng, good assayresults cannot always be obtained because of uneven growth of themicroorganism.

Preferably, the amount of the polyoxyethylene sorbitan fatty acid esterto be incorporated should be in the range of 1 to 20 mg based on 100 mlof the medium for quantitative assay; when the amount exceeds 20 mg, thegrowth of the microorgansim is sometimes inhibited and when the amountis less than 1 mg, uneven growth is noted and good results cannot alwaysbe obtained.

The inoculant that can be used in the present invention may be used bypreincubating the bacteria used in the liquid medium, collecting thebacteria and washing, as in conventional microbiological methods; theinoculant may also be used without preincubation, by collecting thebacteria grown on agar in a conventional manner and washing. It is alsoa characteristic of the present invention that the quantitative assaycan be performed in a simpler manner.

In the quantitative assay method of the present invention, no growth ofbacteria is observed with a variety of substances that lack B₁₂ activityfor example, yeast extract that contains DL-methionine which is an aminoacid, thymidine which is one of nucleic acid components,β-aminolevulinic acid which is a precursor of biosynthesis, cobalt saltswhich are constituents of B₁₂, various amino acids, peptides, vitaminsand nucleic acids, which do not contain B₁₂, etc.; growth of thebacteria is observed with meat extract containing B₁₂. From such it isclear that B₁₂ in a containing substance, 3.g., meat extract, can bespecifically determined quantitatively according to the assay method ofthe present invention.

The microorganism used in the present invention can be dried whilemaintaining a live state and the composition of the dry bacteria can bestored over a long period of time while the microorganism is in a livestate. Accordingly, when the viable bacteria are dried and stored, thedry composition can be readily suspended in a medium solution orsterilized water upon quantitative assay for the B₁₂ -containingsubstance and the bacteria suspension can be used directly as aninoculated bacteria solution for quantitative assay. Further, the liquidculture solution for quantitative assay can be aseptically stored over along period of time, sealed in, for example, an ampule, a glass bottle,etc., stored, unsealed depending upon necessity and provided for assayas a set or kit together with the composition of the dry bacteria forinoculation described above. For this reason, the composition of thebacteria and a predetermined amount of the medium for quantitative assaycomprising pyrroloquinoline-quinone and the polyoxyethylene sorbitanfatty acid ester can be provided as an assay device; when assaying theB₁₂ -containing substance quantitatively using the reagent, it can beaccomplished in an extremely simple manner without requiring storage andcontrol of the bacterial strain. Further, an automated bioassay devicecan be constructed by incorporating the bacteria composition and themedium for quantitative assay in accordance with the present inventioninto the device, respectively to quantitatively assay the B₁₂-containing substance automatically. To dry the viable bacteria, asolution of 10 g of sodium chloride, 20 g of sodium glutamate and 0.2 gof magnesium sulfate in 1 liter of M/30 phosphate buffer is used as adispersion medium and the washed viable bacteria is suspended in thedispersion medium followed by freeze drying. It is advantageous that themedium for quantitative assay be stored and supplied as an ampulebecause such is free from evaporation and dissipiation of methanol. Thebacteria are resistant to salts, grow using methanol as only one carbonsource and are insensitive to compounds other than B₁₂ so that evenextremely rough operation can give accurate analytical data.

Next the present invention will be described by referring to theexamples.

EXAMPLE 1

A medium for storing a bacterial strain was prepared as follows. To 1liter of water were added 2 g of ammonium sulfate, 0.3 g of magnesiumsulfate, 1 g of potassium primary phosphate, 5.5 g of potassiumsecondary phosphate, 30 g of sodium chloride, 0.5 g of meat extract and15 g Of Agar. After sterilizing at 120° C. for 5 minutes, 5 ml ofmethanol was added before agar was solidified. The system was separatelypoured in sterilized test tubes, which were slanted and solidified tomake a slant, respectively. An assay medium was prepared by sterilizing1 liter of a medium for quantitative assay having a composition shown inTable 1 at 120° C. for 5 minutes, adding 2.5 ml of methanol thereto andseparately pouring the medium for quantitative assay into an L-shapedtube by 9 ml each.

                  TABLE 1                                                         ______________________________________                                        Component    Medium A (/l) Medium B (/l)                                      ______________________________________                                        Ammonium     1       g         1     g                                        sulfate                                                                       Magnesium    0.3     g         0.3   g                                        sulfate                                                                       Potassium    2       g         2     g                                        primary                                                                       phosphate                                                                     Potassium    7       g         7     g                                        secondary                                                                     phosphate                                                                     Sodium       30      g         30    g                                        chloride                                                                      Pyrrolo-     --                300   ng                                       quinoline-                                                                    quinone                                                                       "Tween 80"   --                100   mg                                       ______________________________________                                    

As samples for analysis, "ERENTAL" or parenteral nutrient manufacturedby Ajinomoto Co., Inc., sardine meat and mackerel meat were used. 10samples of 1 g each were taken and immersed in 10 ml of deionized water,respectively. After homogenization, heat treatment was conducted at 100°C. for 30 minutes in a hot water bath. The treated liquids werecentrifuged and each supernatant was appropriately diluted. Thedilutions were made test sample solutions.

Alteromonas thalassomethanolica YK 4007 FERM P-1401 was inoculated onthe slant described above. After incubation at 30° C. for a day, thesystem was stored at 5° C. Upon use, the bacteria grown on the slant wassuspended in Medium A and Medium B described above which containedmethanol. After once rinsing with the same medium, respectively,dilution was performed in the same medium solution in such a manner thatabsorbancy (OD) became 0.3 at 610 nm. Thus, bacteria solutions forinoculation were obtained. A standard cyanocobalamin solution and testsample solutions were charged by 1 ml each into L-shaped tubes chargedwith 9 ml each of the aforesaid Medium A and Medium B containingmethanol. Further 0.2 ml of the aforesaid bacteria solution forinoculation was added thereto followed by shake culture at 35° C. for 15hours. The absorbancy of the culture solution was measured as an ODvalue at 610 nm to prepare a standard curve, based on which the amountof cyanocobalamin corresponding to the OD value of the test sample wasdetermined. The results are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________           Methanol-Containing                                                                        Methanol-Containing                                              Medium A     Medium B                                                         Mean         Mean                                                             Value                                                                              Maxi-                                                                             Mini-                                                                             Value                                                                              Maxi-                                                                             Mini-                                                   of 10                                                                              mum mum of 10                                                                              mum mum                                                     Samples                                                                            Value                                                                             Value                                                                             Samples                                                                            Value                                                                             Value                                            Sample (ng/g)                                                                             (ng/g)                                                                            (ng/g)                                                                            (ng/g)                                                                             (ng/g)                                                                            (ng/g)                                           __________________________________________________________________________    ERENTAL                                                                               8.5  9.0                                                                              8.0 8.7   8.9                                                                              8.6                                              Sardine                                                                              55.3 59.8                                                                              50.1                                                                              53.8 54.3                                                                              52.9                                             meat                                                                          Mackerel                                                                             10.2 10.9                                                                              9.3 9.8  10.1                                                                              9.7                                              meat                                                                          __________________________________________________________________________

EXAMPLE 2

The amount of cyanocobalamin contained in "ERENTAL" was quantitativelyassayed in a manner similar to Example 1 except that a medium shown inTable 3 was used as a basal medium, a medium for quantitative assaysupplemented with pyrroloquinoline-quinone having a concentration shownin Table 4 and "Tween 80" was used and "ERENTAL" or parenteral nutrientas used in Example 1 was used.

                  TABLE 3                                                         ______________________________________                                        Medium for quantitative assay:                                                ______________________________________                                        KH.sub.2 PO.sub.4       1.0    g                                              K.sub.2 HPO.sub.4       3.0    g                                              (NH.sub.4).sub.2 SO.sub.4                                                                             1.0    g                                              MgSO.sub.4.7H.sub.2 O   0.3    g                                              NaCl                    30.0   g                                              NaS.sub.2 O.sub.3.5H.sub.2 O                                                                          10.0   mg                                             ZnSO.sub.4.7H.sub.2 O   30.0   μg                                          FeSO.sub.4.7H.sub.2 O   5.0    mg                                             Methanol                5.0    ml                                             Water                   1000   ml                                             pH 6.8                                                                        ______________________________________                                    

The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Addition Concentration                                                        Pyrrolo-          Mean Value                                                  quinoline-                                                                            Tween     of 10      Maximum Minimum                                  quinone 80        Samples    Value   Value                                    (ng/100 ml)                                                                           (ng/100 ml)                                                                             (ng/100 ml)                                                                              (ng/100 ml)                                                                           (ng/100 ml)                              ______________________________________                                        0       0         8.5        9.0     8.0                                      8       0         8.5        8.9     8.0                                      10      1         8.5        8.6     8.4                                      30      10        8.5        8.6     8.4                                      60      20        8.5        8.6     8.4                                      75      20        6.2        6.4     6.0                                      ______________________________________                                    

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for the quantitative assay of vitaminB₁₂ in a substance containing vitamin B₁₂ comprising:adding marinemethanol-utilizing bacteria having vitamin B₁₂ auxotrophy to a mediumfor quantitative assay of said bacteria containingpyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acidester; adding a standard dilution of vitamin B₁₂ in a serialconcentration to a first portion of said medium to culture said bacteriain said first portion; measuring the absorbancy of said first portion asstandard dilution OD values to determine the degree of growth of saidbacteria as a function of vitamin B₁₂ concentration; preparing astandard linear curve of absorbancy versus B₁₂ concentration based onsaid measurements; adding a test substance to a second portion of saidmedium to culture said bacteria in said second portion; measuring theabsorbancy of said second portion as a test substance OD value todetermine the degree of growth of said bacteria; and comparing the testsubstance OD value with said standard linear curve to quantitativelydetermine the B₁₂ concentration in said test substance.
 2. A method asin claim 1, wherein said medium contains 10 to 60 mg ofpyrroloquinoline-quinone per 100 ml of medium and 1 to 20 mg ofpolyoxyethylene sorbitan fatty acid ester per 100 ml of medium.
 3. Amethod as in claim 1, wherein said bacteria is Alteromonasthalassomethanolica YK
 4007. 4. A quantitative assay reagent kit fordetermining the quantity of vitamin B₁₂ in a substance containingvitamin B₁₂ comprising a first vial having sealed therein a dry viablebacteria composition of marine methanol-utilizing bacteria having B₁₂auxotrophy for inoculation; a second vial having sealed therein a mediumfor quantitative assay of said bacteria containingpyrrolo-quinoline-quinone and a polyoxyethylene sorbitan fatty acidester; and a third viral having sealed therein a standard dilution ofvitamin B₁₂ having a serial concentration.
 5. A kit as in claim 4,wherein said medium contains 10 to 60 mg of pyrroloquinoline-quinone per100 ml of the medium and 1 to 20 mg of polyoxyethylene sorbitan fattyacid ester per 100 ml of medium.
 6. A kit as in claim 4, wherein saidbacteria is Alteromonas thalassomethanolica YK 4007.